Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania.

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis, which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis.

Parallel evolution in mosquito vectors - a duplicated esterase locus is associated with resistance to pirimiphos-methyl in An. gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programmes. The organophosphate, pirimiphos-methyl, is a relatively new chemical in the vector control armoury but is now widely used in indoor residual spray campaigns. Whilst generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in Anopheles gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to organophosphates in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in An. gambiae, An. coluzzii and An. arabiensis. As in Cx. pipiens, copy number variation seems to play a role in the evolution of insecticide resistance at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in Anopheles gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programmes.

Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele specific expression.

Malaria control relies on insecticides targeting the mosquito vector, but this is increasingly compromised by insecticide resistance, which can be achieved by elevated expression of detoxifying enzymes that metabolize the insecticide. In diploid organisms, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules (CRM), but few of these have been identified in mosquitoes. We crossed bendiocarb resistant and susceptible Anopheles gambiae strains to identify cis-regulated genes that might be responsible for the resistant phenotype using RNAseq, and cis-regulatory module sequences controlling gene expression in insecticide resistance relevant tissues were predicted using machine learning. We found 115 genes showing allele specific expression in hybrids of insecticide susceptible and resistant strains, suggesting cis regulation is an important mechanism of gene expression regulation in Anopheles gambiae. The genes showing allele specific expression included a higher proportion of Anopheles specific genes on average younger than genes those with balanced allelic expression.

LLIN Evaluation in Uganda Project (LLINEUP) - Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Background: In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus the synergist piperonyl butoxide (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, cross-sectional entomological surveys were carried out at baseline and then every 6 months for two years. In each survey, ten households per HSD were randomly selected for indoor household entomological collections. Results: Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s, but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Conclusions: Distribution of LLINs in Uganda was associated with reductions in parasite prevalence and vector density, but the proportion of infective mosquitoes remained stable, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration:: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae s.l.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of the most widespread tools currently used to control malaria. The genetic underpinnings of resistance are still only partially understood, with much of the variance in resistance phenotype left unexplained. We performed a multi-country large scale genome-wide association study of resistance to two insecticides widely used in malaria control: deltamethrin and pirimiphos-methyl. Using a bioassay methodology designed to maximise the phenotypic difference between resistant and susceptible samples, we sequenced 969 phenotyped female An. gambiae and An. coluzzii from ten locations across four countries in West Africa (Benin, Côte d'Ivoire, Ghana and Togo), identifying single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) segregating in the populations. The patterns of resistance association were highly multiallelic and variable between populations, with different genomic regions contributing to resistance, as well as different mutations within a given region. While the strongest and most consistent association with deltamethrin resistance came from the region around Cyp6aa1 , this resistance was based on a combination of several independent CNVs in An. coluzzii , and on a non-CNV bearing haplotype in An. gambiae . Further signals involved a range of cytochrome P450, mitochondrial, and immunity genes. Similarly, for pirimiphos-methyl, while the strongest signal came from the region of Ace1 , more widespread signals included cytochrome P450s, glutathione S-transferases, and a subunit of the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes were associated with resistance to both insecticide classes, suggesting possible cross-resistance mechanisms. These locally-varying, multigenic and multiallelic patterns highlight the challenges involved in genomic monitoring and surveillance of resistance, and form the basis for improvement of methods used to detect and predict resistance. Based on simulations of resistance variants, we recommend that yet larger scale studies, exceeding 500 phenotyped samples per population, are required to better identify associated genomic regions.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii.

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

A gene expression panel for estimating age in males and females of the sleeping sickness vector Glossina morsitans.

Many vector-borne diseases are controlled by methods that kill the insect vectors responsible for disease transmission. Recording the age structure of vector populations provides information on mortality rates and vectorial capacity, and should form part of the detailed monitoring that occurs in the wake of control programmes, yet tools for obtaining estimates of individual age remain limited. We investigate the potential of using markers of gene expression to predict age in tsetse flies, which are the vectors of deadly and economically damaging African trypanosomiases. We use RNAseq to identify candidate expression markers, and test these markers using qPCR in laboratory-reared Glossina morsitans morsitans of known age. Measuring the expression of six genes was sufficient to obtain a prediction of age with root mean squared error of less than 8 days, while just two genes were sufficient to classify flies into age categories of ≤15 and >15 days old. Further testing of these markers in field-caught samples and in other species will determine the accuracy of these markers in the field.

Negative density-dependent dispersal in tsetse (Glossina spp): An artefact of inappropriate analysis.

Published analysis of genetic material from field-collected tsetse (Glossina spp, primarily from the Palpalis group) has been used to predict that the distance (δ) dispersed per generation increases as effective population densities (De) decrease, displaying negative density-dependent dispersal (NDDD). Using the published data we show this result is an artefact arising primarily from errors in estimates of S, the area occupied by a subpopulation, and thereby in De. The errors arise from the assumption that S can be estimated as the area ([Formula: see text]) regarded as being covered by traps. We use modelling to show that such errors result in anomalously high correlations between [Formula: see text] and [Formula: see text] and the appearance of NDDD, with a slope of -0.5 for the regressions of log([Formula: see text]) on log([Formula: see text]), even in simulations where we specifically assume density-independent dispersal (DID). A complementary mathematical analysis confirms our findings. Modelling of field results shows, similarly, that the false signal of NDDD can be produced by varying trap deployment patterns. Errors in the estimates of δ in the published analysis were magnified because variation in estimates of S were greater than for all other variables measured, and accounted for the greatest proportion of variation in [Formula: see text]. Errors in census population estimates result from an erroneous understanding of the relationship between trap placement and expected tsetse catch, exacerbated through failure to adjust for variations in trapping intensity, trap performance, and in capture probabilities between geographical situations and between tsetse species. Claims of support in the literature for NDDD are spurious. There is no suggested explanation for how NDDD might have evolved. We reject the NDDD hypothesis and caution that the idea should not be allowed to influence policy on tsetse and trypanosomiasis control.

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

Whole-genome sequencing reveals high complexity of copy number variation at insecticide resistance loci in malaria mosquitoes.

Polymorphisms in genetic copy number can influence gene expression, coding sequence, and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single-nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector Anopheles gambiae, caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance. Using the whole-genome sequencing data from 1142 samples in the An. gambiae 1000 genomes project, we identified 250 gene-containing CNVs, encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread (45% of individuals carry at least one of them) and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace. Our detailed descriptions of CNVs found across the species range provide the tools to do so.

Improved spatial ecological sampling using open data and standardization: an example from malaria mosquito surveillance.

Vector-borne disease control relies on efficient vector surveillance, mostly carried out using traps whose number and locations are often determined by expert opinion rather than a rigorous quantitative sampling design. In this work we propose a framework for ecological sampling design which in its preliminary stages can take into account environmental conditions obtained from open data (i.e. remote sensing and meteorological stations) not necessarily designed for ecological analysis. These environmental data are used to delimit the area into ecologically homogeneous strata. By employing Bayesian statistics within a model-based sampling design, the traps are deployed among the strata using a mixture of random and grid locations which allows balancing predictions and model-fitting accuracies. Sample sizes and the effect of ecological strata on sample sizes are estimated from previous mosquito sampling campaigns open data. Notably, we found that a configuration of 30 locations with four households each (120 samples) will have a similar accuracy in the predictions of mosquito abundance as 200 random samples. In addition, we show that random sampling independently from ecological strata, produces biased estimates of the mosquito abundance. Finally, we propose standardizing reporting of sampling designs to allow transparency and repetition/re-use in subsequent sampling campaigns.

Elevated expression of ageing and immunity genes in queens of the black garden ant.

Studies in model organisms have identified a variety of genes whose expression can be experimentally modulated to produce changes in longevity, but whether these genes are the same as those involved in natural variation in lifespan remains unclear. Social insects boast some of the largest lifespan differences known between plastic phenotypes, with queen and worker lifespans differing by an order of magnitude despite no systematic nucleotide sequence differences between them. The contrasting lifespans of queens and workers are thus the result of differences in gene expression. We used RNA sequencing of brains and legs in 1-day-old and 2-month-old individuals of the ant Lasius niger to determine whether genes with queen-biased expression are enriched for genes linked to ageing in model organisms. Because the great longevity of queens may require investment into immune processes, we also investigated whether queen-biased genes are enriched for genes with known roles in immunity. Queen-biased genes in legs were enriched for ageing genes and for genes associated with increasing rather than decreasing lifespan. Queen-biased genes in legs were also enriched for immune genes, but only in 1-day-old individuals, perhaps linked to the changing roles of workers with age. Intriguingly, the single most differentially expressed gene between 1-day-old queen and worker brains was an extra-cellular form of CuZn Superoxide Dismutase (SOD3), raising the possibility of an important role of anti-oxidant genes in modulating lifespan.

Gene expression is more strongly influenced by age than caste in the ant Lasius niger.

Phenotypic plasticity, where a single genome can give rise to different phenotypes, underlies many remarkable features of the natural world and occurs in a wide range of organisms. Understanding the transcriptional differences that underlie plastic phenotypes remains a major unsolved problem in biology. In many ants, females can develop into either queens or workers, two phenotypes with different morphology, behaviour and longevity. In comparison with workers, queens are larger, more fecund and longer lived. Here, we study gene expression differences between queens and workers in the ant Lasius niger. The analysis of age- and tissue-specific RNA sequencing showed that patterns of caste-biased gene expression vary considerably between ages and tissues. Expression was more tightly linked to age than caste despite the important morphological and behavioural differences between queens and workers. Our data allowed us to identify genes that are consistently biased across biological contexts. Caste-biased genes showed faster rates of molecular evolution, lower levels of DNA methylation and greater variability in expression than unbiased genes. Our results indicate that a substantial proportion of caste-biased expression is ephemeral and that taking account of age and tissue is critical to understanding the transcriptomic basis of plastic phenotypes. By contrast, the biological context of expression bias did not broadly affect methylation or the rate of evolution. The faster rate of evolution and greater variability of expression of caste-biased genes indicate that caste-biased genes evolve from loosely regulated genes that can be co-opted for caste-specific tasks because of the lax control over their expression.

Lifespan differences between queens and workers are not explained by rates of molecular damage.

The biological processes that underlie senescence are of universal biological importance, yet they remain poorly understood. A popular theory proposes that senescence is the result of limited investment into mechanisms involved in the prevention and repair of molecular damage, leading to an accumulation of molecular damage with age. In ants, queen and worker lifespans differ by an order of magnitude, and this remarkable difference in lifespan has been shown to be associated with differences in the expression of genes involved in DNA and protein repair. Here we use the comet assay and Western Blotting for poly-ubiquitinated proteins to explore whether these differences in expression lead to differences in the accumulation of DNA damage (comet assay) or protein damage (protein ubiquitination) with age. Surprisingly, there was no difference between queens and workers in the rate of accumulation of DNA damage. We also found that levels of ubiquitinated proteins decreased with age, as previously reported in honeybees. This is in contrast to what has been found in model organisms such as worms and flies. Overall, these results reveal that the link between investment into macromolecular repair, age-related damage accumulation and lifespan is more complex than usually recognised.

Higher expression of somatic repair genes in long-lived ant queens than workers.

Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair.

Ageing and somatic maintenance in social insects.

Social insects offer exciting prospects for ageing research due to the striking differences in lifespan among castes, with queens living up to an order of magnitude longer than workers. A popular theory is that senescence is primarily the result of an accumulation of somatic damage with age, balanced by investment into processes of somatic maintenance. Investigation of these predictions in social insects has produced mixed results: neither damage accumulation nor investment into somatic maintenance is consistently different between castes with different lifespans. We discuss some limitations of the studies conducted thus far and consider an alternative proximate theory of ageing that has been recently proposed.

Caste determination through mating in primitively eusocial societies.

Eusocial animal societies are typified by the presence of a helper (worker) caste which predominantly cares for young offspring in a social group while investing little in their own direct reproduction. A key question is what determines whether an individual becomes a worker or leaves to initiate her own reproduction. In some insects, caste is determined nutritionally during development. In others, and in vertebrate societies, adults are totipotent and the cues that determine caste are less well known. The mate limitation hypothesis (MLH) states that a female's mating status acts as a cue for caste determination: females that mate become reproductives, while those that fail to mate become workers. The MLH is consistent with empirical observations in sweat bees showing that over the course of the nesting season, there are increases in both the proportion of females that become reproductives and the frequency of males in the mating pool. We modelled a foundress's offspring sex-ratio strategy to investigate whether an increasingly male-biased operational sex-ratio over time is evolutionarily stable under the MLH. Our results indicate that such a pattern could occur if early workers were more valuable than late workers. This pattern was then more likely if male mortality was high, if worker mortality was low, if the value of a worker was high and if the period over which workers can help was short. Our results suggest that the MLH can be evolutionarily stable, but only under restrictive conditions. Manipulative experiments are now required to investigate whether mating determines caste in nature.